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Research Article

Achievement of quality standards in HIV testing of a


State Reference Laboratory of North India through
External Quality Assurance Scheme (EQAS): an
evaluation of seven years experience
Banke Lal Sherwal, Sanjib Gogoi, Poonam Gupta, Sarika Suri, Rojalin Nayak, Manoj Jais, Renu Dutta
Department of Microbiology, Lady Hardinge Medical College, New Delhi, India.
Correspondence to: Sanjib Gogoi, E-mail: drgogoisanjib24@gmail.com
Received December 19, 2014. Accepted January 19, 2015

Abstract
Background: Centre for HIV Testing, Lady Hardinge Medical College (CHT-LHMC) is working as a State Reference
Laboratory (SRL) from 2007 and regularly performing External Quality Assurance Scheme (EQAS) activities of

24 Integrated Counselling and Testing Centres (ICTCs) and 16 blood banks (BBs) linked to it.
Objective: To evaluate and analyze data of 7 years (20072013) related to the EQAS activities of SRL (i.e., proficiency
testing and retesting).
Materials and Methods: It was a retrospective study. Proficiency tests were carried out twice a year from 20072013.
The samples from BBs for EQAS retesting were tested using third-generation ELISA kits and samples from ICTCs by the
kits provided by Delhi State AIDS Control Society.
Results: A total of 8194 (4992 samples from BBs and 3202 samples from ICTCs) samples were received over a period
of 7 years for EQAS retesting. HIV-positive samples from BB and ICTCs were 163 (3.26%) and 646 (20.17%), respectively. Nineteen (0.38%) were tested as positive discordant and 3 (0.06%) were tested as negative discordant out of
4992 samples received from the BBs. No indeterminate result was found among the samples from ICTCs. Proficiency
testing showed continuous improvement over the years with 100% participation in years 2012 and 2013. The various attributing factors affecting the test results as well as nonparticipation in proficiency testing were evaluated and
discussed.
Conclusion: CHT-SRL-LHMC is a laboratory accredited by National Accreditation Board for Testing and Calibration
Laboratories, and through EQAS activities the laboratory is providing constant support and opportunities for improvement
of its linked centers.
KEY WORDS: State Reference Laboratory, HIV, External Quality Assurance Scheme, proficiency testing, retesting

Introduction
Quality assurance (QA) refers to planned, step-by-step
activities that let one know that the testing is being carried out
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correctly, results are accurate, and mistakes are found and


corrected to avoid adverse outcome.[1] To achieve high-quality
result, all laboratory personnel in a testing network should be
aware of the procedures for quality performance.[2] According
to the ISO definition, External Quality Assessment Scheme
(EQAS) refers to a system of objectively checking laboratory
results by means of an external agency, including comparison
of a laboratorys result at intervals with those of other laboratories, the main objective being the establishment of trueness.[3]
QA is an integral part of serodiagnosis of HIV as the tests
performed to detect the antibodies against HIV depend on the
quality of the conditions under which the tests are performed,
and thereby, there is always a possibility of false-positive or

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Sherwal et al.: Achievement of an SRL of North India through EQAS

false-negative results. Therefore, consistent reproduction of


reliable test results requires properly maintained QA activities
to control the technical conditions related to performance of
the HIV diagnostic tests.
Two important methods used in EQAS are proficiency
testing (PT) and retesting.
Proficiency testing as per Clinical and Laboratory
Standards Institute guidelines is A program in which
multiple samples are periodically sent to members of a

group of
laboratories for analysis and/or identification;
whereby each laboratorys results are compared with those
of other laboratories in the group and/or with an assigned
value, and
reported to the participating laboratories and
others. It is the most commonly used type of EQA as it
is able to address many laboratory methods. In a typical
PT program, challenge samples are provided at regular
intervals. An optimal frequency will be 34 times yearly.
If the program cannot provide challenges with this
frequency, the laboratory may be able to seek additional
sources. The laboratories participating in the program
analyze the samples and return their results to the central
organization. Results are evaluated and
analyzed, and
the laboratories are provided with information about their
performance and how they compared with other partici
pants. The participating laboratories use the information
regarding their performance to make appropriate changes
and improvements.[4]
Retesting is a program where samples analyzed are
retested, allowing for interlaboratory comparison. It is an
important method in the EQAS of HIV testing. HIV rapid testing presents some special challenges, because it is often
performed outside a traditional laboratory and the kits are
single-use and cannot be subjected to the usual quality control
methods that laboratories use. Therefore, retesting of some
of the samples using a different process such as enzyme
immunoassay (EIA) or ELISA (enzyme-linked immunosorbent
assay) helps to assess the quality of the original testing.
Process of retesting is performed by a reference laboratory to
ensure quality and it is not processed as a blinded process as
it is unnecessary.[5]
Centre for HIV Testing Lady Hardinge Medical College
(CHT-LHMC) is working as a State Reference Laboratory
(SRL) from the year 2007. There are 24 Integrated Counselling and Testing Centres (ICTCs) and 16 blood banks
(10 private and 6 government blood banks) linked to CHTSRL LHMC. CHT-SRL-LHMC conducts EQAS programs as
well as coordinate its linked ICTCs and blood banks (BBs) to
National Reference Centre and conduct regular training programs in collaboration with Delhi State AIDS Control Society
(DSACS) to constantly improve and maintain standard of the
quality of HIV testing done in this centers.
Aims and Objectives
This study was undertaken to retrospectively evaluate the
activities related to EQAS, which include retesting and proficiency testing of linked ICTCs and BBs of SRL from 2007 to 2013.
764

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Materials and Methods


The following activities for QA of HIV testing were
performed as per the NACO guidelines.[6]
EQAS Proficiency Testing
Proficiency testing was not conducted in the year 2007
and 2008, as no panel sera was received from National Reference Laboratory (NRL). In the year 2009, there was only one
round of proficiency testing as SRL had received the panel
sera from the NRL only once, that is, in the month of August
2009. It was in the year 2010 that three rounds of proficiency
testing were conducted. Thereafter, the proficiency testing is
being conducted twice a year. Only the linked ICTCs participated in the proficiency testing, not the BBs. For proficiency
testing, four-member panel sera received from NRL were
divided in aliquots and distributed to each linked ICTC within
7 days of receiving of panel sera from NRL. Representative
from ICTCs come to SRL for collection of proficiency testing
samples. The ICTCs had to perform and send the results of
the proficiency testing samples to SRL within 7 working days
from the date of receiving of proficiency testing samples from
the SRL. The SRL compiled the results of proficiency testing
of all the linked ICTCs and the compiled report was sent to
the NRL within another 7 days of receiving of reports from the
linked ICTCs. Any discordant test results were informed to the
respective centers.
EQAS Retesting
Serum samples were received from linked ICTCs and
BBs from 2007 to 2013 for retesting. The process of retesting
started in the year 2007 with three ICTCs and seven BBs.
These linked centers used to send samples for retesting
every month and the number of samples sent were 5% of
total samples tested in that particular month. This process
continued till end of 2008. From 2009, the process was

streamlined and samples were received on quarterly basis in


the months of January, April, July, and October for retesting.
The linked centers sent 20% positive and 5% negative of the
total samples collected and tested in first 7 working days of
each quarter for retesting.
Samples from ICTCs for EQAS retesting were tested by
the three rapid test kits. The principles of the kits were immunochromatography, immunoconcentration, and ELISA based.
The kits provided by DSACS used to vary, depending on the
availability of the kits, but while performing the rapid testing
of HIV at any point of time the following rules were followed:
(1) the first rapid test kit should have high sensitivity and
the rest two rapid test kits should have high specificity.
(2) The three kits should be of three principles and should
have different antigen preparations.
The samples for retesting from BBs were tested using
third-generation ELISA kits.
Sample rejection criteria were insufficient and hemolyzed
samples, samples not properly labels, and samples not properly sent (three-layer packaging). The results of the samples

Sherwal et al.: Achievement of an SRL of North India through EQAS

retested were sent back to respective centers within 7 days


(turnaround time) of receipt of samples. Few terminologies
used in EQAS retesting are as follows:
Indeterminate result: When a result of the same s ample is
different in ICTC and SRL the sample is termed as
indeterminate.
Discordant result: This term is used for testing of BB EQAS
retesting samples. A sample tested positive for HIV in BB
and negative in SRL is termed as positive discordant (PD)
and a sample tested negative at BB and positive at SRL is
termed as negative discordant (ND) sample.

Results of EQAS Retesting


Blood Banks
A total of 4992 samples were received by SRL-CHTLHMC from 16 linked BBs for EQAS retesting in the past
7 years (20072013). Among them, 163 (3.27%) were
HIV-positive and 4822 (96.59%) were HIV negative; and
22 (0.44%) samples reported discordant results. Nineteen
(0.38%) samples were PD and 3 (0.06%) samples were ND
[Table 2; Figure 1].

Ethical Clearance
This study was based on retrospective collection and
analysis of EQAS activities, which are regularly conducted
by SRL-LHMC under the approval and guidance of National
AIDS Control Organization (NACO), India, under National
AIDS Control Program.

Results
Results of Proficiency Testing
The participation was lowest (40%) in the first round of
proficiency testing in 2011, which showed improvement
(100%) in the second round of proficiency testing in the same
year itself. The detail results of participation of linked ICTCs in
proficiency testing are shown in Table 1.

Figure 1: Result of EQAS retesting of linked blood banks.

Table 1: Result of proficiency testing of linked ICTCs


Years
2009
2010
2011
2012
2013

1st round

84.6% (11/13)
100% (23/23)#
40% (10/22)#
100% (22/22)#
100% (23/23)#
#

2nd round

95.6% (22/23)#
100% (22/22)#
100% (22/22)#
95.8% (23/24)#

3rd round

88% (22/25)#

Total number of ICTCs participated in the proficiency testing/total


number of linked ICTCs at that point of time.
#

Figure 2: Result of EQAS retesting of ICTCs (20072013).

Table 2: Result of EQAS retesting of linked blood banks


Year

2007
2008
2009
2010
2011
2012
2013
Total

Total sample for HIV testing


871
1771
1124
298
253
326
349
4992

HIV positive
26
64
36
13
13
10
12
163

HIV negative
845
1707
1094
285
238
316
337
4822

Negative discordant
0
0
0
0
3
0
0
3#

Positive discordant
0
0
0
4
6
3
6
19

Negative discordant samples: 2 samples (January): first sample (BB tested negative, SRL tested positive, NRL confirmed negative), second
sample (BB tested negative, SRL tested positive, NRL confirmed negative), third sample (July) (BB tested negative, SRL tested positive, NRL
confirmed negative).
#

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Sherwal et al.: Achievement of an SRL of North India through EQAS

Table 3: Result of EQAS retesting of ICTCs


Years
2007
2008
2009
2010
2011
2012
2013
Total

Total samples for HIV testing


156
536
465
529
398
514
604
3202

Total HIV-positive samples


58
230
126
81
44
66
41
646

Total HIV-negative samples


98
306
339
448
354
448
563
2556

No. indeterminate sample


0
0
0
0
0
0
0
0

Table 4: Comparison of results of EQAS retesting of blood banks and ICTCs


Linked center
Blood banks (total no: 16)
ICTC (total no: 24)

HIV
Positive

Negative

Total no of samples
tested for HIV

163
646

4822
2556

4992
3202

ICTC
A total of 3202 were received from the linked ICTCs for
EQAS retesting. Among them, 646 (20.17%) samples were
HIV positive and 2556 (79.83%) samples were HIV negative.
No sample was found to show indeterminate result [Table 3;
Figure 2]. The results of EQAS retesting of BBs and ICTC are
summarized and compared in Table 4.

Discussion
The critical points to the success of HIV and AIDS
programs are the accuracy and reliability of diagnostics and
clinical monitoring of tests performed. With the increasing
disease burden, the expanding HIV testing in developing

countries like India has not kept pace with QA programs to


monitor the performance of these tests, which raises concerns
about test accuracy. To ensure reliability and reduce errors to
a minimum, it is essential to implement a quality system in
all laboratories and testing sites that addresses all aspects of
testing in the laboratory.[3]
In India, the EQAS program has been implemented by
NACO since 2000. It functions with Apex Laboratory and
13 NRL and thousands of subcenters covering the entire
government medical colleges, hospitals , BBs, and primary
health centers in each state. The EQAS program is linked with
the respective State AIDS Control Society in each state for
the effective functioning under the NACO.[2] The first report of
EQAS was published by Belk and Sunderman in 1947.[7]
Discussion of EQAS Retesting Results
A total of 4992 samples from BBs and 3202 samples from
ICTCs were tested in SRL-CHT-LHMC as a part of EQAS
retesting over the period of 7 years (20072013). In the first
3 years, that is, 20072009, the BBs as well as ICTCs used to
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International Journal of Medical Science and Public Health | 2015 | Vol 4 | Issue 6

No. of samples giving discordant/indeterminate results


Positive
discordant

Negative
discordant

Indeterminate

19

send samples for EQAS retesting every month, as described


in the Materials and Method section. Therefore, the number
of samples in that period for EQAS retesting were high
compared to those received from 2010 to 2013. This fact is
reflecting more in the EQAS retesting data of linked BBs.
SRL-LHMC was successful in streamlining the process of
EQAS from later part of 2009 by formulating an EQAS calendar regarding collection of samples for EQAS retesting. This
EQAS calendar was dispatched to every linked center before
starting of EQAS for that particular year. This process has
helped the linked center to understand the process of retesting and send the samples for EQAS retesting in a systematic
and easy manner.
Of 4992 samples from BBs for EQAS retesting, 22(0.44%)
samples gave discordant results [19 (0.38%) PD and
3 (0.06%) ND]. The higher number of PD samples, that is,
the sample tested positive for HIV in the BB and the same
sample tested negative in SRL-LHMC during EQAS retesting, might be attributed to the use of different kits for retesting of samples in BBs and SRL-LHMC. The linked BBs were
using fourth-generation ELISA kits and CHT-SRL-LHMC was
using third-generation ELISA kits for confirmation. Similarly,
the three samples whose results were found to be ND
(sample tested negative in BB and same sample tested positive in SRL-LHMC) were sent to NRL for confirmation and the
final result of all the three samples were given negative by
NRL after conducting Western blot test. Moreover, the use of
the above-mentioned ELISA kits in SRL-LHMC is subjected to
change depending on the availability of the kits at that point of
time. So, there is an urgent need of formulating a new policy
for EQAS retesting of BBs or solving the above-mentioned
discrepancies by uninterrupted supply of similar kits in BBs as
well as SRL for proper comparison and confirmation of EQAS
retesting results, thereby maintaining quality standards of HIV
testing.

Sherwal et al.: Achievement of an SRL of North India through EQAS

In case of EQAS retesting of ICTCs, not a single sample


was tested indeterminate, that is, of 3202 samples tested over
a period of 7 years, the results of the ICTCs and SRL were
similar. This striking difference in results of EQAS retesting
of BBs and ICTCs was because ICTCs are using Rapid Test
kits provided by DSACS for testing and SRL is also using the
same kits (in terms of principles) for confirmation of ICTC
results. This result shows the importance of using similar kits
in all levels of testing and confirmation to minimize the discrepancy of results.
Proficiency Testing
Data reflect that only linked ICTCs are participating in the
proficiency testing, not the BBs. Participation in proficiency
testing is a key component of any laboratory QA program,
whether locally, nationally, or internationally.[4] So, there is a
need of proper guidelines from NACO so as to encourage the
participation of the BBs in the proficiency testing to monitor
the standard of the HIV testing in the linked BBs.
SRL-LHMC started the proficiency testing with 13 linked
ICTCs in 2009. Eleven ICTCs participated in the proficiency
testing in that particular year. The reason of nonparticipation
of rest two ICTCs was nonavailability of the HIV testing kits in
those centers at that point of time. The lowest participation in
proficiency testing was seen in 2011 when only 40% (10/22)
ICTCs participated in the first round of proficiency testing. The
reason of such low participation was also the same, that is, at
11 ICTCs at that point of time, the HIV testing kits were not
available. Therefore, availability of HIV testing kits throughout
the year is of utmost importance so that the linked ICTCs can
participate in both the rounds of proficiency testing to check
their quality standards. To ensure the availability of the kits, it
is the responsibility of the concerned authority to provide them
continuous supply of kits in adequate amount as well as the
responsibility of the laboratory in charge of each linked center to
maintain a proper stock register so as to inform the authority on
time before they exhaust all the kits. From 2012, the participation in proficiency testing showed very encouraging results with
high percentage of participation as mentioned in the Table 1.
The linked ICTCs participating in this proficiency testing gave
100% concordant result from 2009 to 2013 in both the rounds.
Similar kind of studies been performed in different parts
of the world to emphasize and analyze the importance of
proficiency testing. One such extensive study performed in
African region involved the distribution of PT panels to participating laboratories from 2002 to 2010, with a frequency of accurate detection, of anti-HIV-1 and/or anti-HIV-2 antibodies in the
PT panels, ranging from 93% to 100%.[8] Accurate proficiency
testing results are an integral part of laboratory accreditation
process. The importance of proficiency testing procedure
is shown by the fact that, to be recognized by WHO AFRO
(Africa Regional Office), a laboratory must have scored, for
each test, 80% or better on the two most recent PT panels.[9]
The analysis of total 8194 (4992 BB and 3202 ICTC) samples for quality control gave a platform for feedback on the
performance of HIV testing in BBs and ICTCs, which can be of

immense help in taking corrective actions in different factors


affecting the quality of the tests performed. This has also provided an opportunity to find out discordant results that would
otherwise have gone undetected.
Rapid tests with immediate test result are popular in the
USA and Canada. Studies performed in these regions also
emphasize on good laboratory quality control practices, QA,
and participation in HIV proficiency testing to ensure accurate,
timely, and clinically relevant laboratory results.[10]
German Medical Association has given immense stress
in QA in their recent guidelines of diagnosis of infectious
disease. These guidelines are mandatory for all the German
laboratories and Section B2 of the guideline includes QA
of qualitative analyses in laboratory diagnosis of infectious
diseases including HIV.[11]
WHO guidelines for accuracy and reliability of HIV
rapid testing pointed out some limitations of retesting and
proficiency testing, which the laboratories should keep in mind
while conducting retesting and proficiency testing. In case of
retesting of specimens in many countries, there is lack of
capacity at the NRL for retesting the large number of samples and for conducting the needed analysis of data. Long
delays in completing the retesting results in delayed identification of problems. So, WHO has pointed the operational
issues that need to be considered if a retesting program is
to be implemented. Finally, statistical analysis shows that for
low-volume sites, a very large percentage of samples would
have to be retested to detect errors. In proficiency testing for
HIV rapid tests, the panel of specimens sent to the testing
site will not necessarily be tested by all staff, so it is not a
good measure of individual performance. The sample size is
small, so the ability to detect errors is impaired. Also, preparing and distributing specimens for proficiency testing may be
cumbersome for NRLs. Proficiency testing is provided in some
locations, and when available, it is a useful tool in combination
with onsite monitoring.[12]

Conclusion
Established as an HIV testing center in 1992, SRLCHT-LHMC has come a long way and obtained National
Accreditation Board for Testing and Calibration Laboratories
accreditation in 2013. During this phase, it has established
a well-coordinated network through EQAS with its linked
ICTCs, BB, and its mentor NRL [i.e., National Centre for
Disease Control (NCDC), New Delhi]. Apart from conducting
EQAS activities, SRL-LHMC is regularly conducting training
programs for the technicians of the linked centers and help
them to resolve any issues related to testing of HIV. Through
all these activities, SRL-CHT-LHMC is providing constant
support, encouragement, and opportunities for improvement
of its linked centers.
A strong commitment from top-level managers is essential
for the success of the overall quality program. This commitment is important at all levels, and national laboratory leaders
will need to both provide strong leadership at the national
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Sherwal et al.: Achievement of an SRL of North India through EQAS

level and motivate and help laboratory managers throughout the country to understand the system and commit to its
success.

Acknowledgment
We thank all staff working at Centre for HIV Testing (CHT),
Department of Microbiology, Lady Hardinge Medical College,
New Delhi.

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How to cite this article: Sherwal BL, Gogoi S, Gupta P, Suri S,

Nayak R, Jais M, et al. Achievement of quality standards in HIV


testing of a State Reference Laboratory of North India through
External Quality Assurance Scheme (EQAS): an evaluation of seven
years experience. Int J Med Sci Public Health 2015;4:763-768

Source of Support: National AIDS Control Organization


(NACO)/Delhi State AIDS Control Society (DSACS), Conflict of
Interest: None.